Method of sampling and/or depositing a sample of biological matter and device implementing such method

ABSTRACT

The aim of the present invention is a method of sampling all or part of a sample (11) of biological matter (7), which is crude, enriched or cultured through contact with a culture medium (8), such as agar, for example in a Petri dish, using a probe (3) equipped with a terminal end (4), said sampling method comprising the steps of cooling the terminal end (4) of the probe (3), sticking all or part of the sample (11) of biological matter (7) to be sampled through contact, or by applying a pressure exerted by the terminal end (4) onto the sample (11) of biological matter (7), and sampling of all or part of the sample (11) of biological matter (7) so as to separate the sample (11) of biological matter (7) from the culture medium (8), a method of depositing into a container (9) or onto an analysis plate (14) all or part of a sample (11) of biological matter (7) stuck onto a frosted terminal end (4) of a probe, said depositing method including a step of separating the terminal end (4) of the probe (3) from all or part of the sample (11) of biological matter (7), as well as a device (2), a kit and an apparatus (1) implementing these methods.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/808,501, filed Jan. 4, 2013, which is a national stage applicationunder 35 USC 371 of International Application No. PCT/FR2011/051631,filed Jul. 8, 2011, which claims the benefit of French PatentApplication No. 1055565, filed Jul. 8, 2010, the disclosures of whichare hereby incorporated by reference.

The aim of the present invention is a method of sampling and a method ofdepositing a sample of biological matter, crude or enriched culturedthrough contact with a culture medium, as well as a device implementingthese methods.

The sampling of a sample of microorganisms (bacteria, moulds, yeasts orthe like) cultured on an agar culture medium in a Petri dish, or on anyother support, is currently accomplished with the aid of single-usetools such as oeses, sticks, tubes or cones.

However, these consumables do not make it possible to certainly andefficiently sample all types of microorganisms because the latter canhave very different forms, sizes, consistencies, structures orappearances.

On the other hand, these consumables do make it possible to optimallydeposit the biological matter on analysis supports such as plates, norto easily re-suspend said biological matter.

Furthermore, it is important to be able to allow sampling of a bacterialcolony or of a fraction of this colony without sampling the culturemedium situated under the colony: this could vitiate the analysisresults.

The quality of the analysis results also depends on the concentration ofthe deposit of biological matter formed from the sampled sample, and onits homogeneity on the support on which it is deposited.

The present invention aims to remedy all or some of the disadvantagesmentioned above.

To this end, the object of the present invention is a method of samplingall or part of a sample of biological matter, crude, enriched orcultured through contact with a culture medium, possibly agar, using aprobe equipped with a terminal end, said sampling method comprising thesteps of cooling the terminal end of the probe, sticking all or part ofthe sample of biological matter to be sampled through contact of theterminal end onto the sample of biological matter, or by applying apressure exerted by the terminal end onto the sample of biologicalmatter, and sampling all or part of the sample of biological matter soas to separate the sample of biological matter from its support, such asa culture medium.

This method makes it possible to sample any type of sample of biologicalmatter cultured in vitro regardless of form and consistency.Furthermore, with this sampling method, there is no possiblecontamination of the probe because its end, in contact with the sample,can be sterilised after each use, or is never in direct contact with thesample.

Furthermore, with this sampling method, there is not necessarily anyneed to use a single-use terminal end, which reduces the cost of usewhilst allowing faster use cycles (better productivity of the device).

According to one implementation means, the probe is a cryogenic probecomprising means for being cooled.

According to one implementation of the sampling method, prior to thesticking step, a step of wetting the terminal end of the probe in aliquid solution such as distilled water is carried out, in order to forma layer of ice on the terminal end.

This arrangement allows a consumable to be made extemporaneously whichallows the sample to be stuck in order to sample it. Furthermore, withthis implementation of the sampling method, the sample is never directlyin contact with the terminal end of the probe.

According to one implementation of the method, prior to the stickingstep, the sample of biological matter to be sampled is covered with aliquid solution, in order to form a layer of ice around the sampleduring the step of sticking all or part of said sample to the terminalend of the probe.

According to one implementation of the method, the liquid solution istaken from the group comprising water, a saline solution, a buffer, aliquid culture medium or a matrix commonly used for ionising the sampleof biological matter with a view to analysing it using a measuringdevice such as a mass spectrometer. A buffer may for example a carbonatebuffer (10 to 100 mmol/L, ideally 25 mmol/L)

This arrangement makes it possible to reduce by one step the sample massspectrometer analysis method, the matrix having the double function ofbeing used for the sticking step during sampling and for a samplepreparation step with a view to its mass spectrometer analysis.

Alternatively, the liquid solution can be a microorganism culturemedium.

According to one implementation of the sampling method, the step ofwetting the terminal end of the probe or the sample of biological matterto be sampled in a liquid solution is repeated successively at leasttwice, in order to form overlaying layers of ice, like a stalactite.

This arrangement makes it possible to enlarge the sticking surface ofthe stalactite created in this way and thus to be able to sample largersamples.

According to one implementation of the sampling method, the terminal endis removable.

This arrangement makes it possible to considerably increase the usefulsticking surface of the terminal end.

According to one implementation of the sampling method, the terminal endpossesses ferromagnetic properties.

According to one implementation of the sampling method, the step ofsampling all or part of the sample of biological matter consists inapplying a magnetic field, for example using an electromagnet, adjacentto the ferromagnetic terminal end so as to attract the terminal end andthus recover it.

This arrangement makes it possible to automate the sampling withoutrisking the contamination of the sampled sample.

Another object of the present invention is a method of depositing into acontainer or onto an analysis plate all or part of a sample ofbiological matter stuck onto a frosted terminal end of a probe, saiddepositing method including a step of separating the terminal end of theprobe from all or part of the sample of biological matter stuck ontosaid terminal end.

This arrangement makes it possible to employ all or part of the sample.

According to one implementation of the depositing method, the separationstep is carried out without the sample of biological matter stuck on thefrosted terminal end coming into contact with the container or theanalysis plate.

In this case, the step of separating the terminal end from all or partof the sample of biological matter stuck on said terminal end is carriedout by applying a mechanical shock onto the terminal end.

This arrangement makes it possible to immediately collect all of thesampled sample in a frozen state.

According to one implementation of the depositing method, the step ofseparating the terminal end from all or part of the sample of biologicalmatter stuck on the frosted terminal end is carried out by a heatingmeans or in ambient air.

This arrangement makes it possible to collect the sample in a liquidstate and makes it possible to distribute several parts of the sample onseveral different supports.

According to one implementation of the depositing method, the separationstep is achieved by sequential contact of the sample of biologicalmatter with the container or the analysis plate, this contact bringingabout the melting of a superficial layer of ice and the depositing ofbiological matter.

This arrangement makes it possible to obtain control of the quantity ofsample deposited and of the choice of depositing support, and themelting of the superficial layer makes it possible to obtain ahomogeneous deposit, both in terms of distribution of biological matterand the depth of the deposited layer.

According to one implementation of the depositing method, all or part ofthe sample of biological matter is distributed on an analysis plate soas to make several distinct deposits of parts of the same sample ofbiological matter, which is stuck on the terminal end.

This arrangement makes it possible to take several measurements on thedeposits from the same sample, each of these deposits being homogeneous,which makes it possible to improve the quality of certain measurementspectra, in particular for mass spectrometry measurements where a lowdegree of homogeneity in the deposit leads to spectra comprising lessintense peaks with a signal which includes a lot of noise.

According to one implementation of the depositing method, the depositingis effected through continuous contact, for example in the form oflines, of all or part of the sample of biological matter stuck on theterminal end with the container or the analysis plate.

This arrangement makes it possible to achieve a biological matterconcentration gradient on the deposit support, and then to focus themeasures on the gradient zone which makes it possible to obtain the bestresults, for example for mass spectrometry measurements.

Another object of the present invention is a device for sampling anddepositing all or part of a sample of biological matter, crude, enrichedor cultured through contact with a culture medium and intended to bedeposited into a container or an analysis plate, characterised in thatit comprises: a probe equipped with a terminal end, a cooling meansintended for frosting the terminal end, driving means intended to exerta pressure from the probe onto the sample so as to freeze all or part ofthe water contained in the sample in order to stick it to the terminalend, to separate all or part of the sample from its support, such as aculture medium, to bring all or part of the sample to the container orthe analysis plate.

This arrangement provides an automated and reusable device which makesit possible to carry out several sampling and deposition in a minimumamount of time.

According to one embodiment, the device comprises a heating meansintended for detaching the sample from the terminal end.

According to a particular embodiment, the heating means is intended forsterilising the terminal end. Such a sterilisation is carrying outbefore any sampling.

Advantageously, the cooling means and the heating means are composed ofat least one Peltier element. Advantageously, several overlaid Peltierelements are used.

This arrangement allows the sample to pass quicker to liquid state andfacilitates its re-suspension and homogenisation.

According to one embodiment, the terminal end is metallic or mineral.

This arrangement makes it possible to absorb more quickly the heatcontained in the water of the sample, and thus freeze it more quickly.

Advantageously, the terminal end is at least partially covered with ahydrophobic coating or treatment. Such a coating enables easier andoptimised drainage of the liquid and/or the sample present on theterminal end when it is unfrozen.

According to one embodiment, the terminal end is removable.

This arrangement makes it possible to separate the frosted terminal end,in order to stick it directly to the sample of biological matter to besampled.

It furthermore makes it possible to store the terminal end in arefrigerated environment, which can be particularly advantageous if itis desired to carry out an extemporaneous analysis.

According to one embodiment, the terminal end has a shape whichoptimises the sticking of all or part of the sample of biological matterto be sampled or of liquid solution. According to a particularembodiment, the terminal end comprises a pointed end. Advantageously,the shape of the terminal end can be tapered. Such a shape greatlyfacilitates the sticking of the sample or of the liquid solution duringwetting.

This arrangement makes it possible to precisely sample the sample, butalso to sample very small samples such as bacterial microcolonies.

According to one embodiment, the device comprises at least one sensorwhich controls the pressure exerted by the driving means via the probein contact with the sample of biological matter to be sampled which iscultured on the culture medium, and does this in order to halt thispressure.

This arrangement makes it possible to prevent any sticking of the samplesupport, such as an agar culture medium.

According to one embodiment, the sensor is a pressure or force sensor,which can be placed under a Petri dish support for example.

This arrangement makes it possible to control the quantity of biologicalmatter, in the manner of an electronic scale.

According to one embodiment, the device comprises a sensor which detectsthe contact between the terminal end and the biological matter in orderto avoid any pressure.

According to one embodiment, the sensor is a binary electric sensor.

This arrangement makes it possible to prevent any sticking of the samplesupport, such as an agar culture medium, at lower cost.

Another object of the present invention is a kit comprising a device asdescribed previously, which includes a plurality of interchangeableterminal ends.

These terminal ends may or may not be single-use. They may be pointed,loop-shaped, or cylindrical, and of different sizes. This arrangementmakes it possible to have terminal ends of different shapes and sizesfor the same device, in order to adapt to the size of the sample to besampled.

Another object of the present invention is a biological analysisapparatus which contains a device or a kit such as described previously.

In any case, the invention will be better understood with the aid of thefollowing description, with reference to the attached schematic drawingswhich show, in a non-limiting manner, a device which implements thesteps of a method according to the invention.

FIG. 1 shows the overview of a device according to the invention.

FIG. 2 shows some steps of a sampling method according to a firstimplementation according to the invention.

FIG. 3 shows some steps of a sampling method according to a secondimplementation according to the invention.

FIG. 4 depicts several examples of sampling and depositing all or partof a sample.

FIG. 5 depicts an embodiment of a cooling means for the terminal endusing Peltier elements.

FIG. 6 shows a mass spectrum obtained after sampling and depositing acolony of Staphylococcus aureus using the methods according to a firstembodiment of the invention.

FIG. 7 shows a mass spectrum obtained after sampling and depositing acolony of Escherichia coli using the methods according to the firstembodiment of the invention.

FIG. 8 shows a mass spectrum obtained after sampling and depositing acolony of Escherichia coli using the methods according to a secondembodiment of the invention.

As depicted in FIG. 1, a sampling and depositing apparatus 1 accordingto the invention comprises a sampling and depositing device 2.

This sampling and depositing device 2 contains a probe 3, as well asdriving means 5 intended for spatial movement of the probe 3.

This driving means 5 may be constituted by automated articulated arms,or any other equivalent means known to the person skilled in the art.Pressure exerted by the driving means 5 is measured by a sensor (18 a)which is situated below the Petri dish 6 and which controls the descentof the driving means 5, stopping this descent beyond a specifiedpressure threshold.

The probe 3 comprises a metal, removable, pointed terminal end 4, whichcan be replaced by a terminal end 4 with a different-sized point whichmakes it possible to carry out precise samplings on the biologicalmatter 7.

The probe 3, by the action of the driving means 5, moves above a cultureof biological matter 7, constituted here by bacterial colonies culturedon an agar culture medium 8 in a Petri dish 6.

The composition of the biological matter 7 essentially comprises liquidwater.

The probe 3 also comprises a cooling means (not shown) for frosting itsterminal end 4. This means can for example be constituted by liquidnitrogen routed to the terminal end 4 by a conduit situated in the probe3 or by depressurising a refrigerant gas in a volume which is in contactwith the terminal end 4.

It can also be constituted by one or more Peltier elements in contactwith the terminal end 4. In particular, FIG. 5 shows an embodiment inwhich the cooling means comprise two stages 17 a and 17 b of Peltierelements on which the terminal end 4 is mounted.

These elements have the advantage of being used for both cooling andheating the terminal end 4, by reversing their electric supply voltage.

In order to prevent heat exchange with the ambient air, it can beadvantageous to insulate the upper part of the terminal end 4, whichdoes not come into contact with the sample. This insulation makes itpossible to optimise the thermal action of the terminal end 4.

It is in fact possible to envisage having more than two overlaid Peltierelements. The greater the number of elements, the greater the extremetemperatures obtained.

Thus, the cooling and heating of the terminal end 4 will be quicker. Itmay be possible to envisage using this construction to sterilise theterminal end by heating to a high temperature. This is particularlyadvantageous in preventing contamination between the different samplesof biological matter sampled successively.

The cycle of sampling/depositing biological matter 7 can thus besubstantially accelerated and controlled.

The implementation of a sampling method makes it possible to stick tothe terminal end 4 of the probe 3 all or part of the sample 11 of thebiological matter 7 to be sampled; in this present case, a bacterialcolony cultured on the culture medium 8 in a Petri dish 6.

In a first embodiment of the sampling method, a film of ice is depositedon the terminal end 4 by means of the cooling means.

The formation of the film of ice 13 is enabled by the presence of thewater contained in the ambient air.

To this end, the device 2 can directly contain a cryogenic sourceintended for cooling the probe 3 and the terminal end 4.

As shown in FIG. 2, the probe 3 with its terminal end 4, via the drivingmeans 5, is brought into contact with the colony 11 of bacteria 7 whichhas developed on the agar 8 contained in the Petri dish 6.

These driving means 5, via the terminal end 4, apply a pressure on thecolony 11 to be sampled. This pressure associated with the lowtemperature of the film of ice 13 which covers the terminal end 4 makesit possible to freeze the water contained in the bacterial colony 11.

This pressure is measured by a sensor 18 a which is situated below thePetri dish 6 and which controls the descent of the driving means 5,stopping this descent beyond a specified pressure threshold.

Alternatively, the terminal end 4 can simply come into contact with thecolony 11, without exerting pressure on said colony. To do this, it isuseful to have a contact sensor which stops the descent of the drivingmeans 5 once contact is made. Such a sensor may for example be a binaryelectric sensor.

When it freezes, the water contained in the colony 11 forms a solidblock with the layer of ice 13 situated on the surface of the terminalend 4 of the probe 3. All or part of the colony 11 to be sampled isincluded in the ice, and so is stuck onto the terminal end 4.

The pressure or contact exertion time can be advantageously controlledby the device 2.

In a second implementation of the sampling method, depicted in FIG. 3,the terminal end 4 of the probe 3 is wetted in a liquid solution 12,such as liquid water or a matrix commonly used for ionising the sample11 of biological matter 7 with a view to analysing it by a measuringdevice such as a mass spectrometer.

The liquid solution 12 then forms a layer of ice 13 generally in theform of a drop 16 at the end of the terminal end 4. The drop 16 freezesunder the action of the cooling means to form an adhesion surface whichcomes into contact, via the driving means 5, with the bacterial colony11 to be sampled.

In a variant of this second implementation of the sampling method alsoillustrated in FIG. 3, the terminal end 4 of the probe 3 is wetted inthe liquid solution 12 several times in succession in order to increasethe size of the layer of ice 13 used for the adhesion of the colonies 11to be sampled.

It is thus possible to adjust the size of this layer of ice 13 to thedimensions of the colony 11 to be sampled.

In these implementations of the sampling method, it can be advantageousto have a terminal end 4 of the probe 3 which has a shape capable offacilitating the attachment of the drop(s) of liquid solution 12 on saidend. This shape can be tapered. The end can also have a groove, forexample a horizontal groove, on the edge of the end in order to optimisethe attachment of the drop of liquid solution 12.

According to a particular embodiment, the container containing theliquid solution can also have a particular shape. Firstly, it isadvantageous to have a receptacle with a small volume in line with thevolume of the drops(s) to be formed. Secondly, the interior of thecontainer can advantageously have a specified shape correspond to thatwhich the drop must have. Thus, the interior of the receptacle can betapered. It is moreover beneficial for the container to be deep, insofaras it makes it possible to obtain a relatively long frozen drop and thuslimit the risks of contamination of the terminal end 4 by the sample ofbiological matter.

According to another particular embodiment, the container can have itsown cooling means. In that case, the liquid solution 12 contained in thecontainer is cooled. By keeping said liquid solution at a temperatureclose to its freezing point, it is possible to obtain quicker formationof the frozen drop when the terminal end is dipped into the liquidsolution.

In a third implementation of the sampling method (not shown), a drop ofliquid solution 12 is deposited onto the colony 11 to be sampled, suchthat the latter is totally or partially covered in liquid. The terminalend 4 of the probe 3 is then applied onto the drop of liquid solution 12such that the latter is frozen, trapping all or part of the colony 11 tobe sampled and in turn freezing the latter.

In a subsequent step, the driving means 5 separate all or part of thecolony 11 from the agar layer 8, thus accomplishing the sampling step.

The driving means 5 make it possible to move the sampled colony 11 abovea container 9, such as a test tube 9 or an analysis plate 14, such as ananalysis plate used in mass spectrometry.

The implementation of a depositing method subsequently makes it possibleto separate the frosted terminal end 4 of the probe 3 from all or partof the colony 11 stuck onto it.

In this first implementation of this depositing method, the separationbetween the colony 11 and the terminal end 4 is accomplished withoutcontact with any support in ambient air after interruption of the actionof the cooling means. Indeed, once the action of the cooling means isinterrupted, the ice formed on the terminal end 4 of the probe 3 melts,thus freeing the colony which is stuck to it. It then forms a drop, witha more or less substantial volume depending on the quantity of iceformed initially, in which the bacteria are suspended.

In a variant of this first implementation of the depositing methodaccording to the invention, a heating means 10 contributes to making theice holding the colony 11 against the terminal end 4 melt more quickly.

This heating means 10 can equally well be constituted by a flame, a hotair welding unit, convergent light rays, the joule effect produced bypassing a current into a resistive electrical component, or any otherequivalent heat-producing means.

This heating means 10 can also be used subsequently as a sterilisingmeans to sterilise the terminal end 4 of the probe 3.

In the same manner as for the first implementation of the depositingmethod described above, the colony 11 sampled from the secondimplementation of the sampling method described above is also separatedfrom the terminal end 4, by means of a heating means 10.

In this implementation of the depositing method, the colony 11 can alsobe separated from the terminal end 4 of the probe 3 by applying amechanical shock onto the layer of ice 13.

This mechanical shock causes the layer of ice 13 to break. This is thencollected at the same time as the colony 11 sampled in a test tube 9.Such a tube can then be employed for example by an automated analysissystem such as the VITEK® 2 automated system, or an analysis plate 14.

According to a second implementation of the depositing method, all orpart of the colony 11 is deposited through contact of all or part of thecolony 11 onto the analysis plate 14, with this contact progressivelymelting the superficial layer of ice 13 of the sample by the action onthe layer of ice 13 of the surface tension of the surface of thecontainer 9 or of the analysis plate 14.

This contact may be sequential in the form of spots distributed on theanalysis plate 14, or continuous in the form of lines.

Depositing in the form of spots makes it possible to obtain a pluralityof different deposits from the same sample of sampled biological matter(colony 11). These spots can then be employed for example by a massspectrometer.

Depositing in the form of continuous lines makes it possible to obtain aconcentration gradient of biological matter 7 with strongerconcentrations at the start of depositing than at the end of depositing.It is thus possible to take measurements on different zones of theconcentration gradient, in order to locate the zone where the analysisresults are best, when the mass spectrometer is acquiring measurements.

FIG. 4 illustrates some means of implementing the sampling anddepositing methods.

In an application 1A corresponding to the sampling method, the entiretyor a part of the bacterial colony 11 is directly frozen and then sampledin its Petri dish 6 by the probe 3 to then be deposited in a test tube 9in which the bacteria will be in suspension after liquefaction of theicicle formed by the probe 3 around its terminal end 4, with a view toundergoing a conventional analysis.

In an application 1B, part of the bacterial suspension contained in thetest tube 9 is sampled to be deposited onto the analysis plate 14.

In an application 1C, part of the bacterial suspension contained in thetest tube 9 is sampled to be deposited onto another support with a viewto a second conventional analysis, for example a test for sensitivity toantibiotics.

In an application 2A, the bacterial colony 11 or a part of this colonyis directly frozen and then sampled in the Petri dish 6.

A part of this sampling is deposited directly onto the analysis plate 14of a mass spectrometer, whilst another part, in an application 2B, isdeposited onto another support with a view to a second conventionalanalysis, for example a test for sensitivity to antibiotics.

The sampling can also be preserved in frozen form on the probe 3 inorder to defer the second analysis. This is facilitated if the terminalend 4 is removable. In fact, the terminal end 4 can be preserved infrozen form by depositing this end inside a freezer.

The analysis plate 14 used to accomplish the mass spectrometry analysisof all or part of the colony 11 contains a matrix 15 used to ionise thesample.

In a particular implementation, the matrix 15 is in the form of a drieddeposit, after distribution in the form of spots on the analysis plate14.

The liquefaction of the frozen deposit of all or part of the colony 11in contact with the analysis plate 14 makes it possible to re-suspendthe elements of the matrix 15 in a homogeneous liquid mixture alsocontaining the bacteria.

Another solution, illustrated by the application 3A, consists insampling the colony 11 in the Petri dish 6 after wetting the terminalend 4 of the probe 3 directly into the matrix 15 which is in its normalform in liquid state, forming an icicle of matrix 15 around the terminalend 4 of the probe 3.

The liquefaction of the frozen deposit in contact with the analysisplate 14, composed of all or part of the colony 11 sampled by the probe3 and of the matrix 15, will re-suspend the elements of the matrix 15 ina homogeneous liquid mixture also containing the bacteria.

In these two latter embodiments it is important to ensure in advancethat the concentration in elements of ionisation matrix will besufficient with regard to the number of bacteria present in the mixture.

Furthermore, the colonies 11 sampled by these methods, using a non-toxicliquid solution such as water, and reseeded on a Petri dish 6 grow againand are therefore not killed by these sampling and depositing methods.

Examples of industrial applications are suggested below.

EXAMPLES Example 1

In this first example, an ice tip is used to sample an unknownmicroorganism colony, and to deposit it on a mass spectrometer target inorder to identify the colony of unknown microorganisms using a massspectrometer in accordance with the following protocol:

sample the colony on blood agar (bioMérieux ref.) with an ice needle,

with the ice tip, deposit the colony on a 384-position mass spectrometertarget (Bruker). The deposit is obtained by briefly applying the ice tiponto the target at ambient temperature. A slight film of water andmicroorganisms is thus deposited onto the surface of the target.

deposit 2 μl of matrix (alpha-cyano acid dissolved to 10 mg/ml in a50/50 solution of acetone and water) at the surface of the sample.

introduce the target into a MALDI-TOF (Ultraflex II, Bruker) massspectrometer,

adjust the mass spectrometer to optimise the acquisition of massesbetween 2000 and 20000 Da. The person skilled in the art is especiallyaccustomed to adjusting the tension and the laser power of theinstrument.

calibrate the mass spectrometer with the benchmark proteins(ProteinMixte, Bruker).

analyse the sample using 20 series of 50 laser shots. The mass spectrumobtained for each series is summed to obtain the mass spectrum of themicroorganism. The number of shots can be adjusted by the person skilledin the art to obtain the most informative spectrum possible, i.e. withthe most possible and the best defined mass peaks,

determine the masses observed on the mass spectrum of the microorganismusing the Flex Analysis software (Bruker),

compare the masses observed on the mass spectrum of the microorganismwith the masses contained in a database. Several databases exist forthis purpose: the applicant has its own database, and the databases ofthe companies Bruker (Biotyper) or Anagnostec (Saramis) can also beused.

identify the microorganism which has the masses closest to thoseobserved on the mass spectrum of the unknown microorganism.

For this example, this protocol has been applied to two differentcolonies and has made it possible to obtain the mass spectra visible onFIGS. 6 and 7.

The mass spectrum of FIG. 6 has made it possible to identify the colonyas being a Staphylococcus aureus colony.

The mass spectrum of FIG. 7 has made it possible to identify the colonyas being an Escherichia coli colony.

This method is advantageous because it makes it possible to accomplishthe sampling and depositing very quickly with an excellent rate ofsuccess. From the first attempt, all of the colonies, regardless oftheir type, are successfully sampled and then deposited by the iceneedle.

Furthermore, since the target is at ambient temperature, the ice needlemelts very slightly when it touches the surface. The slight stream ofwater which results from this carries the sample and provides a fine andhomogeneous deposit of microorganisms.

This latter point is particularly advantageous because a thick depositcauses signal suppression in mass spectrometry. This phenomenon, whichis well known, is due to the excess of salts and a proportion of matrixwhich is unsuited to the quantity of sample. A heterogeneous deposit isalso disadvantageous, as the signal becomes heterogeneous, which makesit difficult to adjust the mass spectrometer.

Example 2

In a second example, the same protocol is implemented as in example 1,with the difference being that in step 2 the deposit is obtained byrubbing the ice tip on the surface of the mass spectrometer target.

The spectrum figuring in FIG. 8 is obtained. This spectrum leads to theidentification of Escherichia coli.

This protocol has the same advantages as example 1. It furthermore makesit possible to deposit over a larger surface, which can be useful foraccomplishing several successive acquisitions of the same sample, or forsuccessively acquiring several mass spectra with different parameters.

Example 3

In a third example, 25 strains of known species are cultured in a Petridish with a COS (Columbia sheep blood medium, bioMérieux, reference43041) or SDA (Sabouraud glucose medium, bioMérieux, reference 43555)culture medium in accordance with the indications figuring in table 1below:

TABLE 1 BioMérieux BioMérieux culture microor- Culture medium ganismmedium reference Microorganism species reference COS 43041 Burkholderiamultivorans 0301039 COS 43041 Proteus vulgaris 0509142 COS 43041Pseudomonas putida 0509108 COS 43041 Streptococcus pseudopneumoniae0507105 COS 43041 Bacillus licheniformis 0608016 COS 43041 Micrococcusluteus 0602045 COS 43041 Staphylococcus haemolyticus 0704061 COS 43041Proteus mirabilis 0805068 COS 43041 Enterococcus raffinosus 0903030 COS43041 Pseudomonas aeruginosa 1006028 COS 43041 Escherichia coli 1006021COS 43041 Bacteroides fragilis 1009220 COS 43041 Shigella flexneri7709005 COS 43041 Streptococcus pyogenes 7701086 COS 43041 Streptococcusconstellatus 7811150 ssp constella COS 43041 Bacillus megaterium 8004066COS 43041 Chryseobacterium indologenes 8105051 COS 43041 Vibrioparahaemolyticus 8305091 COS 43041 Citrobacter farmeri 8608073 COS 43041Aeromonas hydrophila 606019) COS 43041 Salmonella ser. gallinarum8703202 (pullovorum) COS 43041 Corynebacterium jeikeium 9203007 COS43041 Pseudomonas oryzihabitans 9510157 COS 43041 Enterococcuscasseliflavus 9710016 SDA 43101 Geotrichum capitatum 9409060

After 16 hours of culturing, for each Petri dish, a colony is sampledwith an ice needle and deposited onto a MALDI-TOF target according tothe method of the invention. This operation is repeated to obtain twoindependent deposits for each species studied. The samplings and thedeposits are carried out in accordance with the following operatingmode.

A metallic tip (terminal end), which is cylindrical with a tapered base,kept at −10° C. by Peltier effect (metallic tip in contact with twoPeltier elements in accordance with FIG. 5). It is then dipped for 15seconds into a receptacle of water kept at 9° C.

The metallic tip is removed from the water. A water drop naturallysticks to the metallic tip. This water drop freezes in 15 seconds bytransfer of cold from the metallic tip at −10° C. An ice needle is thusformed.

The needle is positioned on a colony of microorganisms and isimmediately withdrawn. The colony of microorganisms instantly adheres tothe ice needle and remains stuck on the ice needle. The colony ofmicroorganisms is thus sampled by the ice needle. Conversely, the agarof the culture medium is not picked up.

The colony of microorganisms is deposited onto a disposable 48-positionFleximass DS target from Shimadzu. Each depositing position is a circlewith a diameter of 3 mm. Depositing is carried out by rubbing the icepoint on the surface of one of the 48 deposit positions of the targetwith a spiral motion. This motion is initiated at the centre of thedepositing position and completed after 4 circuits so as to cover theentire depositing position with a slight film of water andmicroorganisms.

The sample of microorganisms is dried in open air for several minutes.

1 μL of matrix (alpha-cyano-4-hydroxycinnamic acid ready to be used insolution (bioMérieux, reference 411071) is deposited onto the surface ofthe sample.

The sample of microorganisms and the matrix are dried in open air forseveral minutes.

The target is introduced into a MALDI-TOF mass spectrometer (AximaAssurance, Shimadzu) with a FLEXIMASS DS target support (Shimadzu).

The mass spectrometer is adjusted to optimise the acquisition of massesbetween 2000 and 20000 Da. The person skilled in the art is especiallyaccustomed to adjusting the tension and the laser power of theinstrument.

The mass spectrometer is calibrated with a deposit of E. coli (ATCC 8739strain).

The sample is analysed using 100 series of 5 laser shots. The massspectrum obtained for each series is summed to obtain the mass spectrumof the microorganism. The number of shots can be adjusted by the personskilled in the art to obtain the most informative spectrum possible,i.e. with the most possible and the best defined mass peaks.

the masses observed on the mass spectrum of the microorganism aredetermined with the aid of the LaunchPad version 2.8 software(Shimadzu),

The masses observed on the mass spectrum of the microorganism arecompared with the masses contained in the Saramis (bioMérieux) databaseusing the Spectral ID version 1.1.0 interface (bioMérieux), and themicroorganism is identified by comparison with the mass spectra ofmicroorganisms present in the database.

For this example, the protocol has made it possible to identify thedeposits according to the results set out in table 2 below:

TABLE 2 Deposit No. 1 Deposit No. 2 Probability Probability ExpectedSpecies identified with of Species identified with of microorganism theprotocol from identification the protocol from identification speciesexample 3 (%) example 3 (%) Burkholderia Burkhol. multivorans 100Burkhol. multivorans 100 multi vorans Proteus vulgaris Proteus vulgaris100 Proteus vulgaris 99.99 Pseudomonas Ps. putida 99.99 Ps. putida 99.99putida Streptococcus no identification — Str. pseudopneumoniae 65.67pseudopneumoniae Bacillus B. licheniformis 100 B. licheniformis 99.99licheniformis Micrococcus luteus Mic.luteus/lylae 99.99 Mic.luteus/lylae 100 Staphylococcus Staph.haemolyticus 99.99 Staph.haemolyticus 99.99 haemolyticus Proteus mirabilis Proteus mirabilis 100Proteus mirabilis 100 Enterococcus Entero. raffinosus 99.99Entero.raffinosus 100 raffinosus Pseudomonas Ps. aeruginosa 99.99 Ps.aeruginosa 99.99 aeruginosa Escherichia coli Esch. coli 99.99 Esch. coli99.99 Bacteroides fragilis Bac. fragilis 99.99 Bac. fragilis 99.99Shigella flexneri Esch. coli 99.99 Esch. coli 99.99 Streptococcus Str.pyogenes 100 Str. pyogenes 99.99 pyogenes Streptococcus Str.constellatus 99.9 no identification — constellatus ssp constellaBacillus megaterium B. megaterium 99.99 B. megaterium 99.99Chryseobacterium Chryse. indologenes 99.99 Chryse. indologenes 99.99indologenes Vibrio V. parahaemolyticus 100 V. parahaemolyticus 99.99parahaemolyticus Citrobacter farmeri Citro. farmeri 99.99 Citro. farmeri99.99 Aeromonas Aer. salm. salmonicida 99.99 or Aer. salm. salmonicidaor 99.99 or hydrophila or Aer. hydro./caviae 99.73 Aer. hydro./caviae98.16 Salmonella ser. Salmonella group 78.25 Salmonella group 78.15Galfinarum (pullovorum) Corynebacterium Coryn. jeikeium 100 Coryn.jeikeium 100 jeikeium Pseudomonas Ps. oryzihabitans 99.99 Ps.oryzihabitans 99.99 oryzihabitans Enterococcus Entero. casseliflavus 100No identification — casseliflavus Geotrichum G. capitatum 100 G.capitatum 100 capitatum

The species identified corresponds exactly to the species expected,taking into account the comments below:

Spectral ID provides the abbreviated name of the species. The personskilled in the art is accustomed to these abbreviations. By way ofexample, it is clear to him that G. capitatum means Geotrichumcapitatum, that Entero. casseliflavus means Enterococcus casseliflavus,and so on.

Micrococcus luteus is identified as Mic. luteus/lylae, i.e. as possiblybeing Micrococcus luteus or Micrococcus lylae. It is not possible todifferentiate between these two species using MALDI-TOF analysis and theSaramis database.

Shigella flexneri is identified as Esch. coli, i.e. Escherichia coli. Asabove, Shigella flexneri and Escherichia coli are two species which aretoo close to be distinguishable by MALDI-TOF. Spectral ID and Saramisclass these two species as Escherichia coli.

Aeromonas hydrophilia is identified as Aer. salm. salmonicida orAerhydro./caviae. The 3 species are very close and are difficult todifferentiate by MALDI-TOF.

Salmonella ser. Gallinarum (pullovorum) is identified as Salmonellagroup. MALDI-TOF analysis does not have sufficient resolution tounequivocally distinguish serovar Gallinarum (pullovorum) from the otherserovars. The Spectral ID software and Saramis therefore give only oneidentification at the level of the Salmonella genus.

The protocol has thus made it possible to identify all of the analysedspecies. 72 deposits out of 75 have been identified, which represents97.3% correct identification. This identification rate is very good,indeed even better than the person skilled in the art is accustomed toobtain with manual sampling and deposition followed by a MALDI-TOFanalysis.

This method is advantageous because it makes it possible to accomplishthe sampling and depositing very quickly with an excellent rate ofsuccess and for any type of microorganism (bacteria or yeast (G.capitatum)). In particular, this method makes it possible to sample, anddeposit with equal effectiveness, microorganisms which have verydifferent forms and consistencies (Proteus, Bacillus, coliforms, etc.).

Although the invention has been described in connection with particularimplementation examples and embodiment examples of the invention, it isclear that it is by no means limited by these and that it encompassesall of the equivalent techniques of the steps and means described, andcombinations thereof.

The invention claimed is:
 1. A device (2) for sampling and depositingall or part of a sample (11) of biological matter (7), which is crude,enriched or cultured through contact with a semi-solid culture mediumsuch as an agar medium (8), and intended to be deposited into acontainer (9) or onto an analysis plate (14), comprising: a probe (3)equipped with a pointed and closed terminal end (4), a cooling meansintended for frosting the terminal end (4), driving means (5) intended:for exerting a pressure from the probe (3) onto the sample (11) so as tofreeze all or part of the water contained in the sample (11) in order tostick it to the terminal end (4), for separating all or part of thesample (11) from the culture medium (8), to bring all or part of thesample (11) to the container (9) or the analysis plate (14) and at leastone contact sensor which stops the descent of the driving means (5) oncecontact between the terminal (4) and the biological matter (7) is made.2. The device (2) according to claim 1, wherein the sensor is a binaryelectric sensor.
 3. The device (2) according to claim 1, comprising aheating means (10) intended for detaching the sample (11) from theterminal end (4).
 4. The device (2) according to claim 3, wherein theheating means (10) is also intended for sterilising the terminal end(4).
 5. The device (2) according to claim 1, wherein the cooling meansand the heating means are composed of at least one Peltier element. 6.The device (2) according to claim 1, wherein the terminal end (4) ismetallic or mineral.
 7. The device (2) according to claim 1, wherein theterminal end (4) is at least partially covered with a hydrophobiccoating or treatment.
 8. The device (2) according to claim 1, whereinthe terminal end (4) is removable.
 9. The device (2) according to claim1, wherein the terminal end has a shape which optimises the sticking ofall or part of the sample (11) of biological matter (7) to be sampled orof the liquid solution (12).
 10. A kit comprising a device according toclaim 1 including a plurality of interchangeable terminal ends (4).